Three dimensional analysis of endoplasmic reticulum-Golgi transport machinery by immuno-electron microscopy.

通过免疫电子显微镜对内质网-高尔基体运输机制进行三维分析。

• 批准号: 14580704
• 负责人: YAMAMOTO Akitsugu
• 金额: $ 2.62万
• 依托单位: Nagahama Institute of Bio-Science and Technology (2003-2004)Kansai Medical University (2002)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580704/
• 关键词: 3D immuno-electron microscopy; p125; endoplasmic reticulum; Golgi apparatus; vesicular traffic; spermatid; GM130; microsomal aldehyde dehydrogenase; 3次元免疫電子顕微鏡法; 免疫電顕法; 3次元; 銀増感法; 立体観察

We developed three dimensional immunoelectron microscopy for studying endoplasmic reticulum (ER)-Golgi transport machinery, and applied this technique as fol o s.1. 3D observation of spermatid Golgi apparatus. In spermatids of the Golgi stage, Golgi apparatus develops and participates in the formation of acrosomes. GM130, a cis Golgi protein was detected using colloidal gold-silver enhancement technique, EM sections of 250 nm in depth were observed under an electron microscope at 125kV. Then 3D structure of the Golgi apparatus was analyzed by electron microscopic tomography. As the results, it was clearly shown that GM130 is localized on cytosolic surface of the Golgi apparatus.2. Retention of the ER membrane protein. To study the retention mechanism of an ER membrane protein; microsomal aldehyde dehydrogenase (msALDH), we express GFP protein with retention signal of msALDH at its C-terminal in culture cells. By examining the precise localization of the chimeric protein, it was shown that this protein was excluded from transport vesicle to the Golgi apparatus, and retained in the ER.3. A new protein participating in budding of COPII vesicles from ER. We found a new protein, p125 which binds to a COPII coat protein Sec23, and showed that p125 is localized on the exit sites of the ER and participating in the formation of transport vesicle from ER (Collaboration with Professor Mituo Tagaya, Tokoyo University of Pharmacy and life Science.)3. Using colloidal gold-silver enhancement technique improved in this project, we studied dynamics of the Golgi matrix proteins after a block of the ER-Golgi traffic (Collaboration with Associate Professor Nobuhiro Nakamura, Kanazawa University), accumulation of type IV collagen in the ER in Hsp47 knock-out mice (Collaboration with Professor Kazuhiro Nagata, Kyoto university), and fusion of ER membrane by BNIP1 an apoptosis related protein(Collaboration with Professor Mituo Tagaya, Tokoyo University of Pharmacy and life Science.)

我们开发了三维免疫电子显微镜来研究内质网（ER）-高尔基体运输机制，并按如下方式应用该技术：1。精子细胞高尔基体的 3D 观察。在高尔基阶段的精子细胞中，高尔基体发育并参与顶体的形成。采用胶体金银增强技术检测顺式高尔基体蛋白GM130，在125kV电镜下观察250nm深度的电镜切片。然后通过电子显微镜断层扫描分析高尔基体的 3D 结构。结果清楚地表明GM130定位于高尔基体胞质表面。 2． ER 膜蛋白的保留。研究内质网膜蛋白的保留机制；微粒体醛脱氢酶 (msALDH)，我们在培养细胞中表达 GFP 蛋白，并在其 C 端保留 msALDH 信号。通过检查嵌合蛋白的精确定位，表明该蛋白被排除在高尔基体的运输囊泡之外，并保留在 ER.3 中。一种参与内质网 COPII 囊泡出芽的新蛋白质。我们发现了一种新蛋白 p125，它与 COPII 外壳蛋白 Sec23 结合，并表明 p125 位于 ER 的出口位点，并参与 ER 转运囊泡的形成（与东京药科大学 Mituo Tagaya 教授合作）和生命科学。）3．使用本项目中改进的胶体金银增强技术，我们研究了 ER-高尔基体交通阻断后高尔基体基质蛋白的动态（与金泽大学 Nobuhiro Nakamura 副教授合作），以及 ER 中 IV 型胶原蛋白的积累Hsp47 敲除小鼠（与京都大学 Kazuhiro Nagata 教授合作），以及细胞凋亡相关蛋白 BNIP1 与 ER 膜的融合（与京都大学 Kazuhiro Nagata 教授合作）东京药学生命科学大学田谷三雄教授。）

项目成果

Post-translational targeting of a tail-anchored green fluorescent protein to the endoplasmic reticulum.

尾部锚定的绿色荧光蛋白翻译后靶向内质网。

• 发表时间: 2003
• 期刊: J.Biochem. 134
• 作者: Marutani T;Yamamoto A;Nagai N;Kubota H;Nagata K.;Nakajima k;Masaki R
• 通讯作者: Masaki R

Dynamics of Golgi matrix proteins after a block of ER to Golgi transport

内质网到高尔基体运输受阻后高尔基体基质蛋白的动态

• 发表时间: 2004
• 期刊: J Biochem. 135
• 作者: Yoshimura S;Yamamoto A;Misumi Y;Sohda M;Barr FA;Fujii G;Shakoori A;Ohno H;Mihara K;Nakamura N.
• 通讯作者: Nakamura N.

Masaki R, Kameyama K, Yamamoto A: "Post-translational targeting of a tail-anchored green fluorescent protein to the endoplasmic reticulum."J.Biochem.. 134. 415-426 (2003)

Masaki R、Kameyama K、Yamamoto A：“尾锚定绿色荧光蛋白翻译后靶向内质网。”J.Biochem.. 134. 415-426 (2003)

Dynamics of Golgi matrix proteins after a block of ER to Golgi transport.

内质网至高尔基体运输受阻后高尔基体基质蛋白的动态。

• 发表时间: 2004
• 期刊: J.Biochem. 135
• 作者: Shimoi W;Ezawa I;Nakamoto K;Uesaki S;Gabreski G;Aridor M;Yamamoto A;Nagahama M;Tagaya M;Tani K.;Shimoi W;Yoshimura S
• 通讯作者: Yoshimura S

Accumulation of type IV collagen in dilated endoplasmic reticulum leads to apoptosis in Hsp47-knockout mouse embryos through the induction of CHOP

IV 型胶原在扩张的内质网中的积累通过诱导 CHOP 导致 Hsp47 敲除小鼠胚胎细胞凋亡

• 发表时间: 2004
• 期刊: J Cell Sci 117
• 作者: Marutani T;Yamamoto A;Nagai N;Kubota H;Nagata K.
• 通讯作者: Nagata K.